Metal electrode based 3D printed device for tuning microfluidic droplet generation frequency and synchronizing phase for serial femtosecond crystallography

ABSTRACT

Methods and systems are provided for serial femtosecond crystallography for reducing the vast amount of waste of injected crystals practiced with traditional continuous flow injections. A micrometer-scale 3-D printed water-in-oil droplet generator device includes an oil phase inlet channel, an aqueous phase inlet channel, a droplet flow outlet channel, and two embedded non-contact electrodes. The inlet and outlet channels are connected internally at a junction. The electrodes comprise gallium metal injected within the 3-D printed device. Voltage across the electrodes generates water-in-oil droplets, determines a rate for a series of droplets, or triggers a phase shift in the droplets. An external trigger generates the droplets based on the frequency of an XFEL utilized in droplet detection, thereby synchronizing a series of droplets with x-ray pulses for efficient crystal detection. The generated droplets can be coupled to an SFX with XFEL experiment compatible with common liquid injector such as a GDVN.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a PCT international application and claims benefitof U.S. Provisional Patent Application No. 62/509,538, filed on May 22,2017, and claims the benefit of U.S. Provisional Patent Application No.62/630,105, filed on Feb. 13, 2018, both of which are herebyincorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R21 GM095583awarded by the National Institutes of Health and under 1231306 awardedby the National Science Foundation. The government has certain rights inthe invention.

TECHNICAL FIELD

The present invention relates to systems and methods for performingserial femtosecond crystallography (SFX), and to systems and methods fortuning droplet generation frequency.

BACKGROUND OF THE INVENTION

Proteins operate to perform most of the work of living cells and areinvolved in every cellular process. For example, proteins can replicateand transcribe DNA, and can produce, process, and secrete otherproteins. Proteins not only control cell division and metabolism, butalso oversee the flow of material and information into and out of thecell.

A protein's structure may be characterized to understand how itfunctions. A common method of protein structure determination utilizesX-ray crystallography. Recent advancements have led to serialfemtosecond crystallography (SFX). SFX is a method of X-raycrystallography that uses an X-ray free electron laser (XFEL) toirradiate a protein crystal with a femtosecond pulse of high energyphotons in order to obtain a diffraction pattern before the proteincrystal is destroyed. This diffraction-before-destruction techniquerequires many crystals to be injected into the path of the laser inorder to obtain a complete dataset for constructing an electron densitymap of a protein crystal's structure. The pulse structure of currentlyavailable XFELs ranges from 10 Hz up to 120 Hz. However, conventionalsample delivery methods that use continuous liquid injectors result in alarge amount of wasted protein crystals between laser pulses. SFX canalso be carried out with other pulsed x-ray sources, where pulsedurations surmount the fs time scale.

SUMMARY OF THE INVENTION

Sample waste resulting from the slow pulse frequency of x-ray freeelectron lasers (XFELs) remains a critical issue for serial femtosecondcrystallography (SFX) of proteins. Protein crystals are cumbersome toobtain in suspensions of adequate concentration and large volumes (>1mL) for full datasets. Current XFELs function at pulse frequencies up to120 Hz at LCLS, 60 Hz at SACLA, and 30 Hz at PAL-XFEL, or pulse trainsof 10 Hz frequency at the new European XFEL. Delivering the preciousprotein crystals by a continuous stream is a highly inefficient process,since most of the crystals are not hit by the femtosecond x-ray pulses.

In some embodiments, a system is provided that utilizes a microfluidicdroplet generator coupled to a nozzle to reduce the volume of samplerequired to collect a full data set for SFX experiments with XFELs. Bygenerating small water-in-oil droplets through a microfluidic shearingprocess at a frequency synchronized with the XFEL, the system/methodreduces the amount of sample volume required for a full SFX data setwhen compared to a traditional GDVN alone.

In some embodiments, instead of controlling a phase adjustment to bemade at the point of droplet generation, the invention decouples dropletfrequency from the phase of the droplets. Furthermore, instead of usingactive methods (e.g., piezoelectric, acoustic, etc.) to adjust thephase, some embodiments of the invention use a passive approach with acontinuous introduction of sacrificial oil. In some embodiments, themethod can be applied at existing XFEL facilities around the world andwill also be applicable for newer generation XFELs.

By decoupling droplet generation frequency from the droplet phaseadjustment, more freedom is available to adjust droplet generationconditions. This flexibility translates to more tenability with dropletsize and channel geometries. This ability to adjust these parameters canresult in reduced sample consumption and a decreased risk of clogging,both of which depend on the protein crystal system being studied. Thus,in some embodiments, the invention provides a method that is widelyapplicable to different sizes of protein crystals.

In some embodiments, a system is provided for performing serialfemtosecond crystallography (SFX). A microfluidic T-junction, upstreamof an in-line water-in-oil droplet generator, is utilized to synchronizethe phase of the water-in-oil droplets in a continuous stream with thephase of pulses of a downstream x-ray free electron laser by adding asacrificial oil phase to the sample delivery line.

In some embodiments, the T-junction interfaced between the upstreammicrofluidic droplet generator and the downstream x-ray free electronlaser (XFEL) facilitates adjustment of the flow rate of the oil phasefor water in droplet streams by adding additional oil at a ratecounteracting the phase lag between water droplets and the XFEL laser. Asplitting junction before the T-junction allows for fine flow ratecontrol (order of 0.01 μL/min), below what a typical pressure source(e.g., an HPLC pump) can provide. Downstream of the T-junction is adetector for real-time feedback of the droplet phase.

In some embodiments, a 3-D printed microfluidic device tunes or adjustswater-in-oil droplet generation frequency. Gallium metal basednon-contact metal electrodes embedded in the 3-D printed device are usedto induce local electric fields that change the water-in-oil interface.Fabrication of the gallium metal in the 3d printed device is simple anddoes not require complex fabrication tools and steps. In some systems,tunable frequency ranges in droplet generation by the device mayincrease 10-fold from an original generation frequency.

In some embodiments, a method for fabricating a water-in-oil dropletgenerator device comprises 3-D printing a water-in-oil droplet generatorincluding an oil phase inlet channel, an aqueous phase inlet channel, adroplet flow outlet channel, and a metal inlet, wherein the oil phaseinlet channel, the aqueous phase inlet channel, and the droplet flowoutlet channel are connected at a junction within the droplet generatordevice. Conductive material is injected in the metal inlet to form twonon-contact electrodes embedded in the 3-D printed droplet generationdevice.

In some embodiments, a 3-D printed water-in-oil droplet generator deviceincludes an oil phase inlet channel, an aqueous phase inlet channel, adroplet flow outlet channel, and two embedded non-contact electrodes.The oil phase inlet channel, the aqueous phase inlet channel, and thedroplet flow outlet channel are connected at a junction within thewater-in-oil droplet generator device.

In some embodiments, a system for initiating generation of aqueoussample droplets for serial femtosecond crystallography includes adroplet generator device comprising a micrometer scale 3-D printedwater-in-oil droplet generation system with two embedded non-contactelectrodes, an oil phase inlet channel, an aqueous phase inlet channel,and a droplet flow outlet channel. The two embedded non-contactelectrodes are operable to induce an electric field for initiatinggeneration of an aqueous sample droplet. The system further includes anx-ray free electron laser. A capillary system is connected to thedroplet flow output channel of the droplet generator device. Thecapillary system transmits the aqueous sample droplet in an oil mediumand is positioned to receive an x-ray pulse train from the x-ray freeelectron laser. A droplet detector detects crystal characteristics of acrystal within the aqueous sample droplet transmitted by the capillarysystem when a beam of the x-ray pulse train laser impinges on thecrystal.

In some embodiments, a system for controlling a phase shift andfrequency of aqueous droplets in an oil medium for serial femtosecondcrystallography comprises a droplet generator device comprising metalelectrodes and a T-junction. The T-junction includes an oil phase inletchannel, an aqueous phase inlet channel, and an output channel foroutputting aqueous droplets in an aqueous droplet in oil flow. The metalelectrodes are operable to induce an electric field in the T-junctionfor controlling generation of the aqueous droplets. A droplet detectordetects the aqueous droplets in the aqueous droplet in oil flow based onlaser pulses from an x-ray free electron laser. Droplet generation inthe droplet generator is synchronized with the laser pulses by triggersignals generated across the metal electrodes of the droplet generator.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a functional block diagram of the system for synchronizingdroplets with the operating frequency of an x-ray free electron laser,according to some embodiments.

FIG. 2 is a schematic diagram of the system of FIG. 1, according to someembodiments.

FIG. 3 is a partially transparent overhead view of a frequency and phasedetector of the system of FIG. 1, according to some embodiments.

FIG. 4 is a graph of droplet frequency relative to x-ray pulsesgenerated by the XFEL in the system of FIG. 1, according to someembodiments.

FIG. 5 is a pair of graphs of droplet frequency out-of-phase andin-phase with the XFEL in the system of FIG. 1, according to someembodiments.

FIG. 6A is a partially transparent perspective view of a phasesynchronizer T-junction of the system of FIG. 1, according to someembodiments.

FIG. 6B is a partially transparent overhead view of the phasesynchronizer T-junction of FIG. 6A, according to some embodiments.

FIG. 7 is a block diagram of a control system for the system of FIG. 1,according to some embodiments.

FIG. 8 is a flowchart of a method for synchronizing the droplet flowperformed by the control system of FIG. 7, according to someembodiments.

FIG. 9 illustrates serial femtosecond crystallography (SFX) apparatusand process that utilizes an X-ray free electron laser (XFEL), accordingto some embodiments.

FIG. 10 a flow chart for a SFX droplet generation experiment using anXFEL, according to some embodiments.

FIG. 11 is a perspective cut-away view of a design for amicrometer-scale 3D-printed droplet generator device with metal-basedelectrodes for initiating and tuning droplet generation, according tosome embodiments.

FIG. 12 is a top view and side view of a design for a micrometer-scale3D-printed droplet generator device with metal-based electrodes forinitiating and tuning droplet generation, according to some embodiments.

FIG. 13 includes 1) a top view and side view of a design for amicrometer-scale 3D-printed droplet generator device with metal-basedelectrodes for initiating and tuning droplet generation, 2) an opticalimage of a printed device based on the design, and 3) a description of avariation on the design, according to some embodiments.

FIG. 14 includes 1) a top view and side view of a design for amicrometer-scale 3D-printed droplet generator device with metal-basedelectrodes for initiating and tuning droplet generation, 2) an opticalimage of a printed device based on the design, and 3) a description of avariation on the design, according to some embodiments.

FIG. 15 includes the top view of the design of FIG. 14, a 3-D modelcross-section view of the same design, a 3-D model complete perspectiveview of the same design, a top view of the same design 3-D model, and aside view of the same design 3-D model, according to some embodiments.

FIG. 16 illustrates a fabrication method and procedure including 1)designing and 3-D printing a droplet generator device with metal inletsfor embedded Ga metal-based electrodes, 2) inserting fused silicacapillaries, and 3) injecting Ga metal into the metal inlets of the 3-Dprinted device, and a photo of the device with the Ga filled electrodes,according to some embodiments.

FIG. 17 illustrates a fabrication method and procedure for inserting aplatinum (Pt) wire to connect to a Ga metal electrode in a 3-D printeddroplet generator device, including a top view and top view detail ofthe device, according to some embodiments.

FIG. 18 is a diagram of an experimental setup for generating aqueousdroplets into the flow of an oil medium, and controlling the frequencyof the droplets in the oil by applying an AC or DC potential across theelectrodes of a droplet generator, according to some embodiments.

FIG. 19 includes a side view of a droplet generator device design withmetal electrodes and results of an experiment for on demand dropletgeneration, according to some embodiments.

FIG. 20 includes a side view of a droplet generator device design withmetal electrodes and results of an experiment for on demand dropletgeneration, according to some embodiments.

FIG. 21 represents the results of an experiment including a wettingproperty change in a droplet formation, according to some embodiments.

FIG. 22 illustrates images from results of an experiment utilizing thedevice shown in FIGS. 13, 19 and 20 for tuning droplet frequenciesutilizing an applied potential, according to some embodiments.

FIG. 23 represents a summary of the results of the tuning frequencyexperiment represented in FIG. 22, according to some embodiments.

FIG. 24 illustrates images from the results of an experiment utilizingthe devices shown in FIGS. 13, 19 and 20 for tuning droplet frequencies,according to some embodiments.

FIG. 25 represents the results of an experiment for tuning dropletfrequencies, according to some embodiments.

FIG. 26 represents the results of an experiment for tuning dropletfrequencies, according to some embodiments.

FIG. 27 represents the results of an experiment for a designed 3-Dprinted droplet generation device with Ga metal electrodes, according tosome embodiments.

FIG. 28 illustrates images from experimental results for the dropletgeneration device design of FIG. 27, according to some embodiments.

FIG. 29 illustrates images from experimental results for the dropletgeneration device design of FIG. 27, according to some embodiments.

FIG. 30 represents a summary of experimental results for the dropletgeneration device design of FIG. 27, according to some embodiments.

FIG. 31 illustrates an image from experimental results and a profile forapplied potential for experiments using the droplet generation devicedesign of FIG. 27, according to some embodiments.

FIG. 32 illustrates metal electrode systems for on-demand dropletgeneration by electric triggering, according to some embodiments.

FIG. 33 illustrates methods and systems for on-demand droplet generationby electric triggering, according to some embodiments.

FIG. 34 illustrates four short pulse trigger signals for on-demanddroplet generation by electric triggering, according to someembodiments.

FIG. 35 illustrates thirteen short pulse trigger signals for on-demanddroplet generation by electric triggering, according to someembodiments.

FIG. 36 illustrates droplet phase shift by electric triggering,according to some embodiments.

FIGS. 37 and 38 illustrate a system and method for droplet phaseshifting by electric triggering, according to some embodiments.

FIG. 39 illustrates droplet signal phase shift that is induced by anelectric trigger, according to some embodiments.

FIG. 40 illustrates droplet signal phase shift that is induced by anelectric trigger, according to some embodiments.

FIG. 41 illustrates results of droplet signal phase shifting induced bya manually started electric trigger with a 10 ms pulse width, accordingto some embodiments.

FIG. 42 illustrates results of droplet signal phase shifting induced byan electric trigger with a 100 ms pulse width, according to someembodiments

FIG. 43 illustrates phase shift size differences for droplet signalphase shifting induced by electric triggers with 10 ms and 100 ms pulsewidths, according to some embodiments.

FIG. 44 illustrates droplet signal frequency (Hz) and droplet widths(ms) before and after 10 ms and 100 ms trigger signal pulse widths,according to some embodiments.

FIG. 45 illustrates still images taken from the results of multipleexperiments for determining droplet signal frequencies (Hz) and dropletvolumes generated before and after trigger by a continuous electricsignal pulse of greater than 1 s, according to some embodiments.

FIG. 46 illustrates droplet signal frequencies (Hz) generated by acontinuous electric signal pulse of greater than 1 s for multiplevoltage levels, according to some embodiments.

FIG. 47 is a graph indicting electrowetting effects and the occurrenceof satellite droplets relative to increasing applied potential andincreasing applied frequency, according to some embodiments.

FIG. 48 illustrates an example of a serial femtosecond crystallography(SFX) system using the European Free X-ray Electron Laser (EuXFEL) forapplication of on-demand droplet generation, according to someembodiments.

FIG. 49 illustrates application of on-demand droplet generation byelectric triggering to XFEL or other pulsed X-ray sources, according tosome embodiments.

FIG. 50 illustrates an application of droplet frequency phase shiftingby electric trigger utilizing a gallium electrode device for adjustingdroplet phase, according to some embodiments.

FIG. 51 is a schematic diagram of an electrically triggered dropletworkflow at a EuXFEL beamline experiment station, according to someembodiments.

FIG. 52 includes a photograph of an experimental setup in EuXFEL, an ACtrigger pulse signal, and droplet response, according to someembodiments.

FIG. 53 illustrates a comparison of droplet frequencies before and aftera trigger pulse is applied, according to some embodiments.

FIG. 54 illustrates protein crystal buffer information, according tosome embodiments.

FIG. 55 illustrates a flow diagram for an electrical trigger where alength of the trigger (in time), the initiation of the trigger, anamplitude of an AC trigger signal, an amplitude of a DC trigger signal,and/or a frequency of an AC trigger signal is adjusted dependent on anactual phase delay.

FIGS. 56-76 comprise a series of frames (every 10 frames) captured froma video showing an experiment in operation corresponding to theexperimental results for droplet frequency tuning shown in FIG. 25 andthe experiment device 4-1 shown in FIG. 13.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the following drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are hereby incorporated by reference intheir entirety. The materials, methods, and examples disclosed hereinare illustrative only and not intended to be limiting.

For the recitation of numeric ranges herein, each intervening numbertherebetween with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“About” is used synonymously herein with the term “approximately.”Illustratively, the use of the term “about” indicates that valuesslightly outside the cited values, namely, plus or minus 10%. Suchvalues are thus encompassed by the scope of the claims reciting theterms “about” and “approximately.”

Furthermore, it should also be noted that a plurality of hardware andsoftware based devices, as well as a plurality of different structuralcomponents, may be used to implement various embodiments. In addition,it should be understood that embodiments may include hardware, software,and electronic components or modules that, for purposes of discussion,may be illustrated and described as if the majority of the componentswere implemented solely in hardware. However, one of ordinary skill inthe art, and based on a reading of this detailed description, wouldrecognize that, in at least one embodiment, the electronic based aspectsmay be implemented in software (e.g., stored on non-transitorycomputer-readable medium) executable by one or more processors. As such,it should be noted that a plurality of hardware and software baseddevices, as well as a plurality of different structural components maybe utilized to implement various embodiments. Furthermore, and asdescribed in subsequent paragraphs, the specific configurationsillustrated in the drawings are intended to exemplify embodiments andthat other alternative configurations are possible. For example,“controllers” described in the specification can include standardprocessing components, such as one or more processors, one or morecomputer-readable medium modules, one or more input/output interfaces,and various connections (e.g., a system bus) connecting the components.In some instances, the controllers described in the specification may beimplemented in one of or a combination of a general processor, anapplication specific integrated circuit (ASIC), a digital signalprocessor (DSP), a field programmable gate array (FPGA), or the like.

FIG. 1 illustrates a system and method for controlling droplet formationand delivery for performing serial femtosecond crystallography. Acrystal suspension is mixed with a substrate solution using, forexample, a coaxial mixer (step 1—“Coaxial Mixing”). The mixed sample isthen pinched into droplets by an oil carrier phase (step 2—“DropletGeneration”). Sacrificial oil is then introduced into the droplet lineto synchronize the droplet phase frequency with the x-ray pulsefrequency of the x-ray free electron laser (XFEL) (step3—“Synchronization”). The water-in-oil droplets are injected into thepath of the x-ray by helium focusing gas within a gas dynamic virtualnozzle (GDVN) (step 4—“GDVN”). The droplets are elongated in this jet.

FIG. 2 illustrates the system of FIG. 1 in further detail. An oilreservoir 210, a substrate reservoir 212, and a sample reservoir 214 areeach coupled to a HPLC pump 216 a, 216 b, and 216 c respectively. Thesample fluid from the sample reservoir 214 (e.g., the crystalsuspension) and the substrate solution from the substrate reservoir 212are both pumped into a mixer 218 (e.g., step 1 of FIG. 1). The mixedsolution from the mixer 218 proceeds into the droplet generator 220where it joins a stream of oil pumped from the oil reservoir 210 to formdroplets of the solution in the oil stream (e.g., step 2 of FIG. 1). Thestream of droplets in oil continues to travel downstream through a phasesynchronizer 222 and a droplet detector 224 (e.g., step 3 of FIG. 1 andas described in further detail below) before being expelled through thenozzle 226 (e.g., step 4 of FIG. 1).

The phase synchronizer 222 introduces a stream of sacrificial oil intothe stream of droplets-in-oil in order to adjust the frequency of thedroplets (e.g., the distance between droplets in the stream).Sacrificial oil is controllably pumped from a sacrificial oil reservoir228 by an HPLC pump 230 into a flow splitter 232. From the flow splitter232, part of the stream of the sacrificial oil proceeds to the phasesynchronizer 222 while the rest is diverted into an excess oil reservoir234. The flow splitter 232 enables smaller increases in the sacrificialoil flow rate than the HPLC pump 230 alone, thus increasing theresolution of the phase synchronizer 222.

The introduction of more oil (i.e., the sacrificial oil) into the streamof droplets increases the distance between droplets in the stream. Thestream of droplets then proceeds to the droplet detector 224 that isconfigured to detect the phase changes between oil and droplet todetermine the droplet flow rate (e.g., the time between each new dropletpassing through the droplet detector 224).

FIG. 3 illustrates an example of the droplet frequency and phasedetector 224 in further detail. A NIR laser 310 illuminates atransparent fused silica capillary 312 containing the droplet stream andthe absorbance is detected by a photodetector 314. The absorbance willchange depending on whether oil or droplet is located between the laser310 and the photodetector 314. This change in absorbance results in avoltage change in the output of the photodetector 314 which denotes aboundary of the droplet. The droplet frequency and phase can bedetermined based on the monitored voltage change and, once calculated,can be compared to the XFEL frequency and phase for real timeadjustments of droplet generation and droplet synchronization.

The graphs of FIGS. 4 and 5 illustrate the result of thesynchronization. As discussed above, the voltage output of thephotodetector 314 will change depending on whether a droplet is presentin the detector 314 (i.e., t_(drop)) or oil is present in the detector314 (i.e., t_(oil)). To perform serial femtosecond crystallography (SFX)analysis using the system of FIG. 1, the x-ray pulse of the XFEL wouldbe generated when a droplet is in the x-ray interaction region of theXFEL as shown in the graph of FIG. 4. Both frequency and phase of thedroplet stream are synchronized to the x-ray pulse of the XFEL by thesystem of FIG. 1.

The graph labelled “Out of Phase” in FIG. 5 illustrates an example wherex-ray pulses and droplets in the stream have the same frequency, but areout-of-phase. As illustrated in this example, a droplet will never behit by the x-ray without some adjustment or control. As illustrated inthe graph labelled “In Phase” in FIG. 5, a time shift can adjust thedroplet phase to be in phase with the x-ray pulses of the XFEL. Thistime shift (as well as the frequency of the droplets in the stream) canbe adjusted with the droplet synchronizer (i.e., FIG. 1, step3—“Synchronization”) or the phase synchronizer 222 of FIG. 2.

FIGS. 6A and 6B illustrate and example of a T-junction component 610that can be implemented as the droplet synchronizer in the system ofFIG. 1 (step 3—“Synchronization”). In some implementations, thisT-junction component 610 can also be utilized as the flow splitter 232and/or the droplet generator 220 in the example of FIG. 2. TheT-junction component 610 in this example is a 3D printed component thatincludes two cylindrical channels (an inlet channel 612 and an outletchannel 614) that each gradually taper into a first rectangular channel616. A second inlet channel 618 is also provided that tapers into asecond rectangular channel 620 that couples to the first rectangularchannel 616 to join the main stream. In this example, the width of thefirst rectangular channel 616 is 100 μm while the width of the secondrectangular channel 618 is 75 μm. Both rectangular channels are 75 μmtall. Both of the inlet channels 612 and 618, and the outlet channel 614have a diameter of 380 μm. The entire 3D printed component illustratedin FIGS. 6A and 6B is 1 mm wide, 1.5 mm long, and 0.6 mm high. Thedroplet generator 220 may have the same geometry as shown in FIGS. 6Aand 6B. Thus, this device can be employed both for synchronization ofwater droplets by adding oil to water in oil segmented flow, or togenerate the water droplets in oil. In FIG. 6B, if employed under“synchronization mode” oil would be added to the inlet 618 and thesegmented oil water solution would be delivered to the inlet 612.

FIG. 7 illustrates an example of a control system configured to operatethe SFX system of FIG. 1. A controller 701 includes an electronicprocessor 703 and a non-transitory computer-readable memory 705. Thememory 705 stores instructions that are executed by the electronicprocessor 703 to provide the functionality of the controller 701. Thecontroller 701 is coupled to each of the four pumps 707, 709, 711, 713as illustrated in FIG. 2. The controller 701 is also coupled to thelaser 715 and the photodetector 717 of the droplet detector illustratedin FIG. 3.

FIG. 8 illustrates an example of a method executed by the controller 701of FIG. 7 to adjust the frequency and phase of the droplets in theoutput stream. The controller 701 monitors the output of thephotodetector 717 to determine a frequency and phase of the droplets inthe stream passing through the droplet detector (step 801). Based onthis determined frequency & phase of the droplets and a knownfrequency/phase of the x-ray pulses of the XFEL, the controller 701determines whether the droplet stream has the same frequency and thesame phase as the x-ray pulses (step 803). If the frequency and/or thephase are not synchronized, the controller 701 adjusts the sacrificialoil pump 713 which, in turn, alters the frequency and/or phase of thedroplets in the output stream (step 805). This control loop is repeatedby the controller 701 until the phase and frequency of the dropletstream both match the phase and frequency of the x-ray pulses of theXFEL.

In serial femtosecond crystallography (SFX) experimentation utilizingX-ray free electron laser (XFEL) detection for the investigation ofprotein crystal structures, the volume of wasted sample material betweenlaser pulses may be reduced by synchronizing the protein crystaldelivery rate with the x-ray beam repetition rate.

Methods and systems are provided for fabrication of a 3D-printedwater-in-oil droplet generator. The generator device can initiate andtune droplet generation frequency by inducing local electric fields fromembedded Gallium (Ga) metal-based electrodes in a micrometer-scale3D-printed device. The non-contact gallium electrodes can preventdegradation and damage of biological samples such as precious proteincrystals. These electrodes can also reduce various problems, such asJoule heating, and can avoid hydrolysis. The Ga metal electrode designcan be easily adapted to many different configurations of 3D-printedmicrofluidic devices. These methods do not require complex fabricationprocesses such as deposition or microfabrication methods.

The initiating droplet generation and tuning droplet generationfrequency in a 3D-printed device is achieved by polarizing an oil-waterinterface utilizing an application of alternating current (AC)potential, thereby adjusting interfacial tension to induce dropletbreakup. This active droplet generation 3D-printed device offersincreased control of droplet frequency and droplet size, and possessesthe ability to synchronize the phase of droplet generation. Phasesynchronization is accomplished utilizing an external trigger, such asthat of an X-ray free electron laser used in serial femtosecondcrystallography. Furthermore, the generated droplets may be coupled toan SFX with XFELs experiment compatible with a common liquid injectorsuch as a gas dynamic virtual nozzle (GDVN).

FIG. 9 illustrates serial femtosecond crystallography (SFX) apparatusand process that utilizes an X-ray free electron laser (XFEL). Referringto FIG. 1, a serial femtosecond (fs) crystallography experiment utilizesshort fs XFEL laser pulses that intersect with a liquid sample stream(jet). Front and rear CCD detectors capture the interactions of thesample and the XFEL laser pulses. XFEL lasers currently in use, forexample, LCLS in Stanford, EuXFEL in Germany, PAL-XFEL in Korea, orSACLA in Japan, and a future XFEL laser SwissFEL in Switzerland, pulseat repetition rates of 10 Hz up to 120 Hz. During the time periods whena laser is not pulsing, for example, in between pulses, sample in theinjected liquid stream is wasted, thus wasting precious protein crystalsamples.

The present method and system provides a droplet generator that deliversdroplets of crystal containing solution intersected by an oil phase, andtunes the frequency of the droplets. For example, the frequency of thedroplets may be tuned to a frequency between 10 and 120 Hz, depending onthe XFEL instrument. However, the system is not limited to any specificfrequency range and the system may be tuned to other frequencies.

FIG. 10 is a flow chart for an exemplary SFX droplet generationexperiment using an XFEL. As shown in FIG. 2, pressure applied by anHPLC pump 1010 forces oil to flow into a droplet generator 1012. The oilserves as a medium for carrying droplets of a sample. Another HPLC pump1014 provides pressure to force sample material carried in a liquid, forexample, crystals in an aqueous liquid, to a T junction 1016 in thedroplet generator 1012, where the droplet generator 1012 injectsdroplets of the sample liquid into the oil medium. The sample dropletsin the oil medium flow to a droplet detector 1018 and out of a nozzle1020. Pressure from the HPLC pumps 1010 and 1014 may influence thefrequency of droplets in the flow.

In some embodiments, an electric field is induced within the dropletgenerator 1012 to initiate sample droplet generation, or to vary thefrequency of sample droplets flowing away from the droplet generator1012. For example, the droplet generator 1012 may comprise non-contactconductive electrodes. Direct current (DC) or alternating current (AC)potentials may be applied to the electrodes to induce the local electricfields to modify the frequency of the droplets.

The pump pressures may influence the droplet frequency without theapplied electric potential. Typically, the flow rate ratio of oil towater determines an established droplet frequency. Upon application ofan AC or DC potential, that frequency is altered. The system can also beoperated under constant pressure conditions. In this case, a pressureratio of the oil and water lines determines a “base” frequency. Infurther regime, droplets are not generated under the constant pressuremode, but are initiated via the applied electric potential.

An applied field may cause an electrowetting effect that may change thecontact angle of the oil/water/device interface. This in turn may changethe likelihood of droplet release. At higher frequencies, high speedvideos show an instability (fluctuation) of the oil/droplet interface,which then leads to droplet breakup. At even higher frequencies,dielectric breakdown may happen, spraying many small droplets into theoil phase.

In some embodiments, the droplet generator 1012 comprises a 3-D printeddevice. The electrodes of the droplet generator 1012 may comprisegallium (Ga) metal based non-contact electrodes embedded in the 3-Dprinted device. The embedded electrodes may be fabricated by injectingthe Ga metal into inlets of the 3-D printed device. The embeddedelectrodes may be used to induce the local electric fields that initiatedroplets or tune the frequency of the droplets, and may change thewater-in-oil interface. In some embodiments, the droplets may begenerated based on a trigger for applying a potential across theelectrodes. For example, the trigger may be based on the frequency ofthe XFEL laser. Such a method may be referred to as on demand dropletgeneration.

In some systems, a power source, such as a function generator incombination with a voltage amplifier, are utilized to generate the DC orAC waveforms applied to the droplet generator 1012 electrodes. Forexample, the voltage source devices may be connected via wires andconductive adhesive to the metal electrodes embedded in the 3D printeddroplet generator 1012. See FIG. 10 for an example of an experimentalsetup.

A contact angle of the water/oil/device interface, and its changes, maybe referred to as wetting properties. Electrowetting effects may lead toextension of an aqueous droplet to an opposite device wall or a dropletdragging on a wall of the device. When an electric potential is notapplied in the device, the device walls are hydrophobic and thehydrophilic droplet does not attach or stick to the wall. With anelectric potential applied, the droplet can touch the adjacent devicewall before it releases. This may typically occur at higher frequenciesof applied electric potential.

The device walls may be hydrophobic because they are coated with acoating agent, for example, Novec 1720 (a product of 3M). The devicematerial may be proprietary. A resist may be used in printing. Themeasured the contact angle of the material has been found to behydrophilic. Therefore the device walls are rendered hydrophobic toavoid droplets sticking to the wall in a regular (no potential) dropletgeneration device.

FIG. 11 is a perspective cut-away view of a design example for amicrometer-scale 3D-printed droplet generator device with metal inlets1112 for embedding (injecting) gallium (Ga) metal-based electrodes 1110in the 3D printed device. The electrodes 1110 are utilized to induceelectric fields in the device for initiating droplet generation and/ortuning the frequency of droplet generation.

FIG. 12 is a top view and side view of a design example for amicrometer-scale 3D-printed droplet generator device with metal inlets1212 for embedded GA metal-based electrodes 1210 utilized for initiatingdroplet generation and/or tuning the frequency of droplet generation byinducing local electric fields in the 3D-printed device.

FIG. 13 includes 1) a top view and side view of a device design 4 for amicrometer-scale 3D-printed droplet generator device with metal inlets1312 for embedded Ga metal-based electrodes 1310 utilized for initiatingdroplet generation and/or tuning the frequency of droplet generation byinducing local electric fields, 2) an optical image of a printed devicebased on the design, and 3) a description of a variation 4-1 on thedevice design 4.

FIG. 14 includes a top view and side view of a design for amicrometer-scale 3D-printed droplet generator device. The designincludes metal inlets 1412 for injecting Ga metal-based material to formthe electrodes 1410 that are utilized for initiating droplet generationand/or tuning the frequency of droplet generation by inducing localelectric fields. FIG. 6 further includes an optical image of a printeddevice based on the design, and a description of a variation on thedesign.

FIG. 15 includes the top view of the design shown in FIG. 14, a 3-Dmodel cross-section view of the same design, a 3-D model completeperspective view of the same design, a top view of the same design 3-Dmodel without metal electrode inlets, and a side view of the same design3-D model without metal electrode inlets.

FIG. 16 illustrates a method and procedure for fabricating a dropletgenerator device with metal electrodes, including 1) designing and 3-Dprinting the droplet generator device with metal inlets for injecting Gametal-based electrodes, 2) inserting fused silica capillaries into thedevice ports, and 3) injecting Ga metal into the metal inlets. Alsoshown is a photo of the device with the Ga filled electrodes.

FIG. 17 illustrates a method and procedure for inserting a platinum (Pt)wire to connect to a Ga metal electrode in a 3-D printed dropletgenerator device, including a top view and top view detail of thedevice.

FIG. 18 is a diagram of an experimental setup for generating aqueousdroplets into the flow of an oil medium, and controlling the frequencyof the droplets in the oil by applying an AC or DC potential across theelectrodes 1810 of a droplet generator. The oil phase is forced into anoil capillary 1812 by a pump 1814. The aqueous phase is forced into anaqueous capillary 1816 by a pump (not shown). Aqueous droplets areformed in the oil and flow out of the droplet generator via a thirdcapillary 1818. An electric field is induced by the applied potential,which modifies the rate of droplet generation. Flow rates in the systemare controlled via the external pumps. The rate of generation of theaqueous droplets may be modified depending on the level of the appliedpotential and/or the frequency of the applied potential. For example,the applied electrical field may speed-up droplet generation. In someembodiments, the generation of a droplet may be triggered based on thefrequency of a XFEL laser utilized, for example, in serial femtosecondcrystallography (SFX) apparatus and process that utilizes an X-ray freeelectron laser (XFEL). For example, the applied potential may becontrolled based on the frequency of the XFEL. In some embodiments, afunction generator in combination with a voltage amplifier may generatethe DC voltage or AC waveforms. The power source device may be connectedvia wires and conductive adhesive to the metal in the droplet generator,for example.

FIG. 19 includes a side view of a droplet generator device design withmetal electrodes and results of an experiment for on demand dropletgeneration.

FIG. 20 includes a side view of a droplet generator device design withmetal electrodes and results of an experiment for on demand dropletgeneration.

FIG. 21 represents the result of an experiment including a wettingproperty change in a droplet formation.

Based on experimental results utilizing device design 5, AC potentialscan speed up droplet frequency. Pressure ratios induce a specificdroplet frequency, which can be increased upon application of the ACpotential. The frequency has been enhanced by up to a factor of 10.However, other layouts may provide a greater increase in frequency.Electric wetting phenomena and potentially dielectrophoretic effects maycause the complex response observed. Droplet frequency tuning wasdemonstrated at low pressure <15 psi, but also at higher pressures (˜200psi) compatible with serial femtosecond crystallography experimentalflow rates.

FIG. 22 illustrates images from results of an experiment utilizing thedevice shown in FIG. 13 for tuning droplet frequencies utilizing anapplied potential, according to some embodiments.

FIG. 23 represents a summary of the results of the tuning frequencyexperiment represented in FIG. 22.

Based on experimental results utilizing device design 4, DC potentialscan induce droplets on demand (droplet release in order of seconds). ACpotentials can induce droplets on demand (also slow process).

FIG. 24 illustrates images from the results of an experiment utilizingthe devices shown in FIGS. 13, 19 and 20 for tuning droplet frequencies,according to some embodiments.

FIG. 25 represents the results of an experiment for tuning dropletfrequencies. See also FIGS. 56-76 that comprise a series of framescaptured from a video showing this experiment in operation. Theexperiment was performed on the apparatus 4-1 shown in FIG. 13.

FIG. 26 represents the results of an experiment for tuning dropletfrequencies.

FIG. 27 represents the results of an experiment for a designed 3-Dprinted droplet generation device with Ga metal electrodes.

FIG. 28 represents illustrates images from experimental results for thedroplet generation device design of FIG. 27, according to someembodiments.

FIG. 29 illustrates images from experimental results for the dropletgeneration device design of FIG. 27, according to some embodiments.

FIG. 30 represents a summary of experimental results for the dropletgeneration device design of FIG. 27.

FIG. 31 illustrates an image from experimental results and a profile forapplied potential for experiments using the droplet generation devicedesign of FIG. 27, according to some embodiments.

A system and method is provided for a metal electrode based 3D printeddevice for tuning droplet generation frequency. On demand dropletgeneration by electric triggering produces droplets that are generatedbased on electric trigger signals. Droplet phase may be shifted based onelectric triggering where phase shifting of droplets generationfrequency is tuned by electric trigger signals. Application of thesystem in European Free X-ray Electron Laser (EuXFEL) includes dropletscontaining KDO8 crystals tested for the phase shifting by an electrictriggering method in EuXFEL.

FIG. 32 illustrates metal electrode systems for on-demand dropletgeneration by electric triggering, according to some embodiments. Ametal electrodes device version (5-1) includes a distance of t=10 μm,where t is the distance between the electrode and fluidic channel.Version (5-1) has added void spaces 2300 that reduce printing time atthe corner of the device (green areas). 3D printing time is reduced from12 hours per device to 8 hours per device. The distance t (red arrows)is reduced from 50 μm to 10 μm to reduce the applied voltage. Thetrigger threshold voltage is decreased from 1000 V to 600 V.

FIG. 33 illustrates methods and systems for on-demand droplet generationby electric triggering, according to some embodiments. In someembodiments, aqueous liquid droplets from an aqueous reservoir aregenerated or pumped into an oil liquid from an oil reservoir by shortpulse electric DC signals.

FIG. 34 illustrates four short pulse trigger signals for on-demanddroplet generation by electric triggering, according to someembodiments. Referring to FIG. 34, four short pulses are utilized totrigger the generation of four droplets. For example, a trigger pulsewidth of less than 300 ms may be utilized.

FIG. 35 illustrates thirteen short pulse trigger signals for on-demanddroplet generation by electric triggering, according to someembodiments. Referring to FIG. 35, thirteen short pulses are utilized totrigger the generation of thirteen droplets. For example, a triggerpulse width of less than 200 ms may be utilized.

FIG. 36 illustrates on-demand droplet generation by electric triggering,according to some embodiments. Referring to FIG. 36, a relatively veryshort pulse is utilized to induce a phase shift of the droplet frequencywhere the phase shift trigger signal may be an AC electric signal. Forexample, a droplet triggering signal AC pulse of 10 ms or 100 ms may beapplied where a droplet frequency signal is generated at 11 Hz.

FIGS. 37 and 38 illustrate a system and method for droplet phaseshifting by electric triggering, according to some embodiments.Referring to FIGS. 37 and 38, droplets are generated in a metalelectrode device 3428 including a T junction that receives oil liquidvia an oil line from an oil reservoir 3412 connected to a first HPLCpump 3410 and liquid received via an aqueous line from a samplereservoir 3426 that is connected to a second HPLC pump 3420. The liquidfrom the oil reservoir 3412 passes through a flow sensing system 3414prior to reaching a first inlet of the metal electrode device 3428. Asecond flow sensor 3424 is placed between the second HPLC pump 3420 andthe sample reservoir 3426. Flow sensor data is read by a dataacquisition system (DAQ) 3416 and trigger signals are generated by adigital to analog converter (DAC) 3416, amplified, and sent to the metalelectrode droplet generator 3428. Droplets output from the metalelectrode device 3428 are detected in a droplet detector 3430.

T-split connectors 3422 are used for both of the aqueous (Aq) line andthe oil line to reduce the fluctuation of flow rate originated from theHPLC pumps 3410 and 3420. Split ratio and hydraulic resistance can becalculated and tuned by the length and diameter of a capillary. Thus,droplets generation and generation frequency are consistent and stable.

In some embodiments, the DAQ 3416 and ADC 3416 may include a powerlabsystem and labchart software for measuring the flow rates from the flowrate sensors 3412 and 3424 and droplet signals from the droplet detector3430. Both of flow rates and droplet signals can be detected easily.

The droplet detector 3430 is used for detecting droplet signals and thedroplet generation frequency may be calculated using labchart software.Other calculations such as period of droplets, phase shift, and dropletwidth (volume) can also be calculated using labchart software.

FIG. 39 illustrates droplet signal phase shift that is induced by anelectric trigger, according to some embodiments. An example short pulsetrigger signal of 10 ms causes a phase shift in the droplet signalwithout changing the droplet width and frequency. The droplet width andfrequency do not change (or are approximately the same) before and afterthe trigger signal.

FIG. 40 illustrates droplet signal phase shift that is induced by anelectric trigger, according to some embodiments. A phase shift (t_(p))may be calculated based on a silent time (t_(s)) in a droplet signal andthe average frequency (f_(avg)) of the droplet signal as:t _(p) =t _(s) −n*(1/f _(avg))  (1)where n is the number of droplets that should occur during the silenttime t_(s).

FIG. 41 illustrates results of droplet signal phase shifting induced bya manually started electric trigger with a 10 ms pulse width, accordingto some embodiments. A phase shift of approximately 3 ms to 88 ms isachieved with an electric triggering signal. The droplet width andfrequency do not change before and after the trigger signal. The phaseshift correlates with the start time of the trigger that was initiatedmanually at a random time. The results show the time resolution of themethod down to a few milliseconds. This process can be automated withhardware and software components, similar to what is shown in FIG. 55.

FIG. 42 illustrates results of droplet signal phase shifting induced byan electric trigger with a 100 ms pulse width, according to someembodiments. A phase shift of approximately 3 ms to 88 ms is achievedwith an electric triggering signal. The droplet width and frequency donot change before and after the trigger signal.

FIG. 43 illustrates phase shift size differences for droplet signalphase shifting induced by electric triggers with 10 ms and 100 ms pulsewidths, according to some embodiments. A 100 ms pulse width triggercauses a larger phase shift and larger silent time as compared to a 10ms pulse width trigger. Phase shifts of approximately 3 ms to 88 ms areachieved by electric triggering signal pulse durations of 10 ms and 100ms respectively. There are no changes in droplet width and frequencyseen before and after the trigger signal.

FIG. 44 illustrates droplet signal frequency (Hz) and droplet widths(ms) before and after 10 ms and 100 ms trigger signal pulse widths,according to some embodiments.

FIG. 45 illustrates still images taken from the results of multipleexperiments for determining droplet signal frequencies (Hz) and dropletvolumes generated before and after trigger by a continuous electricsignal pulse of greater than 1 s, according to some embodiments.

FIG. 46 illustrates droplet signal frequencies (Hz) generated by acontinuous electric signal pulse of greater than 1 s for multiplevoltage levels, according to some embodiments. By applying a continuoussignal, the droplet generation frequency is increased up to ×2.5. Above300 V, an electro-spraying effect is observed.

FIG. 47 is a graph indicting electrowetting effects and the occurrenceof satellite droplets relative to increasing applied potential andincreasing applied frequency, according to some embodiments.

FIG. 48 illustrates an example of a serial femtosecond crystallography(SFX) system using the European Free X-ray Electron Laser (EuXFEL) forapplication of on-demand droplet generation, according to someembodiments. SFX using an X-ray free electron laser (XFEL) may be usedto determine protein structures. SFX with XFEL's include resourceintensive methods that require large amounts of protein crystals, forexample, up to 1 g of protein. Gas dynamic virtual nozzles (GDVNs) maybe used to inject a continuous stream of protein crystals into the pathof the XFEL. However, pulsed X-rays such as 120 Hz at LCLS 10 MHz burstsin a 10 Hz train structure at EuXFEL), result in significant wastage ofinjected protein crystals between pulses. Therefore, the frequency ofthe generated droplets should match the pulse structure of the XFEL. Asignificant challenge to this method of reducing sample consumption ismatching the phase of droplet generation with the phase of the X-raypulses.

FIG. 49 illustrates application of on-demand droplet generation byelectric triggering to XFEL or other pulsed X-ray sources, according tosome embodiments. Droplets containing protein crystals and XFEL signalare synchronized to reveal protein crystal structures and to reduce theamount of protein sample crystals required for serial femtosecondcrystallography.

FIG. 50 illustrates an application of droplet frequency phase shiftingby electric trigger utilizing a gallium electrode device for adjustingdroplet phase, according to some embodiments. Droplet generationfrequency and phase can be modulated by electric triggering. A metalelectrode inserted in the droplet generator can improve dropletsynchronization with X-ray pulses. Electric trigger experiments weretested at the EuXFEL, which is located in Hamburg, Germany. A metalelectrode device can be used to trigger the droplets and forsynchronizing the droplet frequency with the XFEL signal by an electrictrigger. The droplet generation frequency and XFEL frequency may besynchronized by causing a phase shift in the generated droplets.

FIG. 51 illustrates a schematic diagram of an electrically triggereddroplet workflow at a beamline experiment station in EuXFEL, accordingto some embodiments. The experiment set-up includes a real-time dropletdetector and an example of a digital readout from the droplet detector.

FIG. 52 includes a photograph of an experimental setup in EuXFEL, an ACtrigger pulse signal trace, and a droplet response, according to someembodiments. Possible AC voltages that are applied for electricaldroplet triggering and frequency tuning include 100V, 200V, 250V and300V, at 300 Hz. Droplet responses confirmed that the droplets weretriggered by the trigger AC pulse signal of the electrical trace.

FIG. 53 illustrates a comparison of droplet frequencies before and aftera trigger pulse is applied, according to some embodiments. Dropletgeneration frequency does not change significantly after the triggerpulse in EuXFEL experiments. Various conditions were applied totriggering of the droplets. Observed frequency (Hz) and droplet width(ms) did not change significantly after the trigger pulse. Thesefindings agree with results achieved at Arizona State University.

FIG. 54 illustrates protein crystal buffer information, according tosome embodiments

FIG. 55 illustrates a flow diagram for an electrical trigger where alength of the trigger (in time) is adjusted or the initiation of thetrigger is adjusted dependent on an actual droplet phase delay. In step5501 a droplet frequency or droplet phase is detected by the dropletdetector 3430, shown in FIG. 37. In step 5503, if the droplet frequencyor phase is not synchronized with the laser of the detector, the DAQ andADC 3416 adjusts the length of the trigger in time, or the initiationtime of the trigger depending on the detected droplet frequency or phaseto synchronize the droplets with the droplet detector's laser. In someembodiments, the amplitude of an applied AC signal, the amplitude of anapplied DC signal, and/or a frequency of an applied AC signal of theelectrical trigger can be adjusted based on the actual droplet phasedelay.

FIGS. 56-76 comprise a series of frames captured from a video showing anexperiment in operation corresponding to the experimental results fordroplet frequency tuning shown in FIG. 25 and the experiment device 4-1shown in FIG. 13. The video frames should be read in rows from left toright, and then from the top of the page to the bottom of the page.

In summary, water-in-oil droplets were created using a 3D-printeddroplet generator with integrated Ga electrodes using two DC short pulsesignals that trigger the generation of droplets (i.e., on-demand dropletgeneration). A short pulse trigger, for example, of 10 ms or 100 mscauses a droplet phase shift with no change in droplet frequency anddroplet width. Also, a long pulse trigger, for example, greater than 1s, induces a droplet frequency increase.

Although the invention has been described in detail with reference tocertain preferred embodiments, variations and modifications exist withinthe scope and spirit of one or more independent aspects of the inventionas described.

What is claimed is:
 1. A 3-D printed water-in-oil droplet generatordevice, the device comprising: an oil phase inlet channel; an aqueousphase inlet channel; a droplet flow outlet channel; and two embeddednon-contact electrodes, wherein the oil phase inlet channel, the aqueousphase inlet channel, and the droplet flow outlet channel are connectedat a junction within the water-in-oil droplet generator device, whereinthe junction includes a linear channel section in which the oil phaseinlet channel is coaxially coupled to the droplet flow outlet channel,wherein the two embedded non-contact electrodes are positioned onopposite sides of the linear channel section of the junction such thatthe two embedded non-contact electrodes are arranged on an axis thatintersects the linear channel section, wherein the aqueous phase inletchannel is connected to the junction at the linear channel section, andwherein the axis between the two embedded non-contact electrodesintersects the linear channel section of the junction at a locationwhere the aqueous phase inlet channel connects to the oil phase inletchannel.
 2. The device of claim 1, wherein the two embedded non-contactelectrodes are positioned in the 3-D printed water-in-oil dropletgenerator device such that a potential applied across the twonon-contact electrodes initiates generation of water-in-oil droplets ordetermines a rate of generation of the water-in-oil droplets.
 3. Thedevice of claim 1, wherein the water-in-oil droplets form at thejunction of the oil phase inlet channel, the aqueous phase inletchannel, and the droplet flow outlet channel.
 4. The device of claim 1,wherein the two non-contact electrodes comprise gallium.
 5. The deviceof claim 1, wherein the 3-D printed water-in-oil droplet generatordevice is a micrometer scale 3-D printed device.
 6. The device of claim1, wherein the water-in-oil droplet generator device initiates dropletgeneration when local electric fields are induced by the two non-contactelectrodes.
 7. The device of claim 1, wherein the water-in-oil dropletgenerator device adjusts droplet generation frequency when localelectric fields are induced by the two non-contact electrodes.
 8. Thedevice of claim 1, wherein water-in-oil droplet generation is triggeredin the water-in-oil droplet generator device based on a frequency of anx-ray free electron laser.